DESCRIPTION: (Applicant's Description) The completion of many genome sequences presents the challenge of understanding how their derived proteome contributes to a living organism. To begin to understand the complex matrix of interactions in a living cell, we propose a pilot project aimed at obtaining a comprehensive map of the protein interactions within the first fully sequenced eukaryote, Saccharomyces. We will combine four newly available technologies to produce a powerful, rapid high-throughput methodology to study protein interactions in the normal cellular context. First, we will develop more versatile variants of our rapid genomic tagging technique, which creates functional protein chimeras expressed at their normal level and carrying a Protein A affinity tag at their C-termini. Second, we will further refine our subcellular fractionation protocols, in order to routinely dissect cells from the tagged strains into fractions enriched in the tagged protein and its specifically interacting partners. Third, we have developed novel and extremely efficient protocols for the immunopurification of the tagged subcomplexes from these different fractions. Fourth, the proteins so isolated will be rapidly identified by mass spectrometry (MS), using both our current and developing techniques. The speed and sensitivity of these MS techniques permits the analysis of large numbers of proteins made from relatively small amounts of cells. For this pilot project, we will select a subset of approximately 200 proteins to analyze, corresponding to approximately 3% of the yeast proteome. The members of this group will be chosen such that they represent the broad spectrum of characteristics found in the proteome, and include proteins with strong interest to the biological community. Each member will be tagged and co-isolated with its specifically interacting partners. We will use these proteins for two purposes. First, we will refine our analytical approach, defining and then eliminating any bottlenecks that we encounter, and extending our methodology to include transient interactions and regulatory modifications of proteins. We will explore the possibilities for automation at every stage, to increase speed and throughput. Second, we will apply the technology to case studies on functionally interrelated subsets of proteins within the group. For a case study in yeast, we will investigate the cell cycle control protein-interaction and modification network occupied by cyclins (universal regulators of the eukaryotic cell cycle). Our case study in humans will be the p53-MDM-2 complex, a key player in preventing oncogenesis. We will analyze the protein interactions and modifications relating to the function of this complex. The resulting wealth of information will be used to construct a pilot functional proteomic database, providing a matrix of functional interactions between proteins within a living cell.